shRNA

Lentiviral Systems

MISSION shRNA
If I want to make my own viral particles, which format should I purchase?

What should I do if I have trouble obtaining high yields of plasmid DNA?

How many cells can I infect with the amount of lentivirus provided?

How does Sigma measure viral titer?

What is the method you recommend for screening the stable clones? Is there a pair of primers that can be used for screening the clones by PCR?

Do you offer any cells for packaging?
Can the MISSION lentiviral particles be further propagated in the lab?

Can I extract the vector directly from the lentiviral particle?

What is the titer for the Lentiviral transduction particles?

Is a titering range of 105 - 106 too low?

How do you determine functional viral titer (TU/ml) using
the p24 assay?


How do the features of the viral system work?

If I want to make my own viral particles, which format should I purchase?
Sigma offers ready-to-use viral particles (200 µl at 1 x 106 TU/ml) to eliminate these steps for you. However, if you choose to make your own viral particles, we recommend the purified plasmid format for packaging protocols that require less than 1 µg of plasmid DNA (i.e. typical protocols for 60mm dishes utilize 1 µg of transfer vector and yield 4-5ml of viral particles). For large-scale production of viral particles, we recommend the bacterial glycerol stocks. The bacterial format allows for propagation of the shRNA transfer vector and facilitates larger scale DNA purification required for scaled up packaging co-transfections.


What should I do if I have trouble obtaining high yields of plasmid DNA?
Generally, high yields of DNA can be achieved with much of the collection. However, viral-based vectors are known to be somewhat difficult to prep. If particular clones are troublesome, we recommend streaking the bacterial stocks on LB/carbenicillin plates to isolate a single colony and DNA purification with GenElute™ Endotoxin free Midi (Product Code NA0200) or GenElute™ Endotoxin free HP Maxi (Product Code NA0400S) prep kits.

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Can the MISSION lentiviral particles be further propagated in the lab?
No, the viral particles cannot be propagated for biosafety reasons. The MISSION TRC lentiviral particles are replication incompetent. The particles are made using features of 2nd and 3nd generation lentiviral packaging systems. Genes for replication and structural proteins are absent in the packaged viral genome since these genes are supplied by other plasmids in the packaging cells. The viral genome contains only the region between the 5’ and 3’ LTRs of pLKO.1. In addition, the lentiviral vector contains a self-inactivating 3’ LTR that renders it unable to produce infectious virus once it integrates into the host chromosome.


How do I know if my cell type can be transduced?
The lentiviral particles are pseudotyped with VSV-G Protein. This allows transduction of a wide range of cell types.


How many cells can I infect with the amount of lentivirus provided?
The amount of cells that can be infected depends upon the cell line being used. For HeLa, A549 and HEK293T cells we have used 4 µl (1 x 106 TU/ml via p24 assay) for each well containing ~16,000 cells in a 96-well plate. Primary or other difficult cells may require more lentiviral supernatant, but even at a 10 µl level, it would give you enough for 20 x 96-well reactions. We suggest decreasing the number of cells plated to increase the multiplicity of infection (MOI) if necessary. Also, performing a limiting dilution titer on your cell line will give you the optimal amount of viral particles needed for each assay.

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Can I extract the vector directly from the lentiviral particle?
No, this is not possible. The vector is used to make the particles in the packaging or producer cells. The viral genome contains only the RNA version of the region found between the 5’ and 3’ LTRs of pLKO.1 (promoter, hairpin sequence, puromycin resistance gene, etc.)


What is the titer for the Lentiviral transduction particles?
Each clone is provided as 200 µl containing approximately 1 x 106 TU/ml (via p24 titering assay).


How does Sigma measure viral titer?
We use a p24 titer assay, which is an ELISA against the p24 viral coat protein.

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Is a titer of 106 too low?
We have optimized our viral production and this titer gives excellent integration efficiency in cultured cells. You may be more familiar with adenovirus, which titers at a range around 109. Lentivirus, however, has many advantages over adenovirus such as stable integration and ability to transduce primary and non-dividing cells, etc. If there are applications that require you to use higher titer virus, the highly stable VSV-G envelope allows concentration of lentiviral particle via ultracentrifugation.


How do you determine functional viral titer (TU/ml) using the p24 assay?
We determine the TU/ml based upon the p24 assay from ZeptoMetrix. We use the conversion from Didier Trono to determine the relationship between pg/ml p24 and viral titer or MOI:

There are approximately 2000 molecules of p24 per physical particle (PP) of lentivirus:
(2 x 103) x (24 x 103 Da of p24 per PP), 48 x 106/Avogadro = (48 x 106) / (6 x 1023) = 8 x 10-17 g of p24 per PP, approximately 1 PP per 1 x 10-16 g of p24, 1 x 104 PP per pg of p24

A reasonably well packaged, VSV-G pseudotyped lentiviral vector will have an infectivity index in the range of 1 TU per 1000 physical particles (PP) to 1 TU per 100 PP (or less). Thus, the range is approximately 10 to 100 TU/pg of p24. It is through this conversion that TU/ml is obtained. Our current viral packaging protocol has given us highly reproducible titers.

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What is the method you recommend for screening the stable clones and is there a pair of primers that can be used for screening them by PCR?
Puromycin resistant clones may be screened via qRT-PCR using primers designed against PAC or the target gene of interest.


Do you offer any cells for packaging?
No, we do not currently offer packaging cells. We recommend the commonly available engineered cell line HEK 293T. It is imperative that the cells you use be healthy, never grown to confluency, etc. for the best viral titers.

How do the features of the viral system work?
The library is lentiviral based. This allows for the production of viral particles in an appropriate packaging cell line. Upon infection with the resulting lentiviral particles, the shRNA sequence is integrated into the chromosome for stable expression of the hairpin RNA. The VSV-G envelope is pantropic and allows delivery to virtually any cell. In addition, lentivirus does not require a mitotic event for integration into the host cell genome.


Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.



MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.

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